In the field of molecular diagnostics, the amplification of nucleic acids from numerous sources has been of considerable significance. Examples for diagnostic applications of nucleic acid amplification and detection are the detection of viruses such as Human Papilloma Virus (HPV), West Nile Virus (WNV) or the routine screening of blood donations for the presence of Human Immunodeficiency Virus (HIV), Hepatitis-B (HBV) and/or C Virus (HCV). Furthermore, said amplification techniques are suitable for bacterial targets such as mycobacteria, or the analysis of oncology markers.
The most prominent and widely-used amplification technique is Polymerase Chain Reaction (PCR). Other amplification reactions comprise, among others, the Ligase Chain Reaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and Qβ-amplification.
Automated systems for PCR-based analysis often make use of real-time detection of product amplification during the PCR process in the same reaction vessel. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels:
It has been shown that amplification and detection of more than one target nucleic acid in the same vessel is possible. This method is commonly termed “multiplex” amplification and requires different labels for distinction if real-time detection is performed.
It is mostly desirable or even mandatory in the field of clinical nucleic acid diagnostics to control the respective amplification using control nucleic acids with a known sequence, for qualitative (performance control) and/or quantitative (determination of the quantity of a target nucleic acid using the control as a reference) purposes. Given the diversity especially of diagnostic targets, comprising prokaryotic, eukaryotic as well as viral nucleic acids, and given the diversity between different types of nucleic acids such as RNA and DNA, control nucleic acids are usually designed in a specific manner.
In an in-vitro-diagnostic environment, assays based on nucleic acid amplification and detection are typically controlled externally, i.e. the respective control nucleic acid is processed in a vessel separate from the target nucleic acid. It can serve as a qualitative or a quantitative external control nucleic acid. In a qualitative setup, the control serves as a positive control for evaluating the validity of the amplification and detection reaction. In a quantitative setting, the control serves, alternatively or additionally, as a reference for determining the quantity or concentration of the target nucleic acid.
Fluid samples derived from biological fluids such as, for instance, human blood, often exhibit a significant complexity. Therefore, in order to mimic the fluid matrix of the biological samples possibly containing one or more target nucleic acids, external control nucleic acids used in in-vitro diagnostics usually have to be provided in essentially the same fluid matrix as said biological samples. For example, for the nucleic acid diagnostic analysis of human plasma, an external control nucleic is generally provided in normal human plasma (NHP, pooled plasma from several healthy donors), especially in order to comply with regulatory requirements (Guidance for Industry and FDA Staff—Assayed and Unassayed Quality Control Material, 2007; Section IV, B, 1. Matrix Effects).
The present invention provides a controlled amplification method using a different approach that displays various advantages.